Glycosyltransferases are enzymes involved in in vivo biosynthesis of sugar chains on glycoproteins, glycolipids and the like (hereinafter referred to as “complex carbohydrates”). Their reaction products, i.e., sugar chains on complex carbohydrates have very important functions in the body. For example, sugar chains have been shown to be important molecules primarily in mammalian cells, which play a role in cell-cell and cell-extracellular matrix signaling and serve as tags for complex carbohydrates during differentiation and/or development.
Erythropoietin, a hormone for blood erythrocyte production, can be presented as an example where sugar chains are applied. Naturally-occurring erythropoietin is disadvantageous in that it has a short-lasting effect. Although erythropoietin is inherently a glycoprotein, further attempts have been made to add new sugar chains onto erythropoietin, as a result of which recombinant erythropoietin proteins with an extended in vivo life span have been developed and produced and are now commercially available. In the future, there will be increasing development of such products in which sugar chains are added or modified, including pharmaceuticals and functional foods. Thus, it is required to develop a means for freely synthesizing and producing sugar chains. In particular, the development of glycosyltransferases is increasing in importance as one of the most efficient means.
Until now, about 150 or more glycosyltransferase genes have been isolated from eukaryotic organisms including humans, mice, rats and yeast. Moreover, these genes have been expressed in host cells such as CHO cells or E. coli cells to produce proteins having glycosyltransferase activity. On the other hand, about 20 to 30 types of glycosyltransferase genes have also been isolated from bacteria which are prokaryotic organisms. Moreover, proteins having glycosyltransferase activity have been expressed in recombinant production systems using E. coli and identified for their substrate specificity and/or various enzymatic properties.
Sialic acid is often located at the nonreducing termini of sugar chains and is therefore regarded as a very important sugar in terms of allowing sugar chains to exert their functions. For this reason, sialyltransferase is one of the most in demand enzymes among glycosyltransferases. As to β-galactoside-α2,6-sialyltransferases and their genes, many reports have been issued for those derived from animals, particularly mammals (Hamamoto, T., et al., Bioorg. Med. Chem., 1, 141-145 (1993); Weinstein, J., et al., J. Biol. Chem., 262, 17735-17743 (1987)). However, such animal-derived enzymes are very expensive because they are difficult to purify and hence cannot be obtained in large amounts. Moreover, such enzymes have a problem in that they have poor stability as enzymes. In contrast, as to bacterial β-galactoside-α2,6-sialyltransferases and their genes, reports have been issued only for those isolated from microorganisms belonging to Photobacterium damselae (International Publication No. WO98/38315; U.S. Pat. No. 6,255,094).
However, Photobacterium damselae-derived β-galactoside-α2,6-sialyltransferase has a productivity of 550 U/L when produced from Photobacterium damselae (Yamamoto, T., et al., Biosci. Biotechnol. Biochem., 62(2), 210-214 (1998)), while the productivity is 224.5 U/L when this β-galactoside-α2,6-sialyltransferase is produced from E. coli cells transformed with plasmid pEBSTA178 carrying its gene (Yamamoto, T., et al., J. Biochem., 123, 94-100 (1998)). Thus, there is a demand for an enzyme having higher productivity. On the other hand, Photobacterium damselae-derived β-galactoside-α2,6-sialyltransferase has a specific activity of 5.5 U/mg (Yamamoto, T., et al., J. Biochem., 120, 104-110 (1996)). In this regard, there is also a demand for an enzyme having higher activity.
Among known bacterial sialyltransferases, Pasteurella multocida-derived α2,3-sialyltransferase can be listed as an enzyme whose productivity and activity are relatively high, although it is categorized as a different type of enzyme. This enzyme has a productivity of 6,000 U/L (Yu, H., et al., J. Am. Chem. Soc., 127, 17618-17619 (2005)) and a specific activity of 60 U/mg.
To meet the high demand of sialyltransferases, there is a need for β-galactoside-α2,6-sialyltransferases having higher productivity and/or activity.
Patent Document 1: International Publication No. WO98/38315
Patent Document 2: U.S. Pat. No. 6,255,094
Non-patent Document 1: Hamamoto, T., et al., Bioorg. Med. Chem., 1, 141-145 (1993)
Non-patent Document 2: Weinstein, J., et al., J. Biol. Chem., 262, 17735-17743 (1987)
Non-patent Document 3: Yamamoto, T., et al., Biosci. Biotechnol. Biochem., 62(2), 210-214 (1998)
Non-patent Document 4: Yamamoto, T., et al., J. Biochem., 123, 94-100 (1998)
Non-patent Document 5: Yamamoto, T., et al., J. Biochem., 120, 104-110 (1996)
Non-patent Document 6: Yu, H., et al., J. Am. Chem. Soc., 127, 17618-17619 (2005)